Using a new imaging technique, National Institutes of Health researchers have uncovered for the first time that the biological machinery that builds DNA can insert molecules into the DNA strand that are damaged as a result of environmental exposures. These damaged molecules trigger cell death that produces several human diseases, according to the researchers.

The findings, published online ahead of print in the journal Nature, provide a possible explanation for how one type of DNA damage may lead to cancer, diabetes, hypertension, cardiovascular and lung disease, and Alzheimer’s disease.

In the study, researchers from the National Institute of Environmental Health Sciences (NIEHS) used time-lapse crystallography — a technique that takes snapshots of biochemical reactions occurring in cells. — to determine that DNA polymerase, the enzyme responsible for assembling the nucleotides, or building blocks, of DNA, incorporates nucleotides with a specific kind of damage into the DNA strand.

That damage, explained senior NIEHS researcher Dr. Samuel Wilson, M.D., is caused by oxidative stress, or the generation of free oxygen molecules, in response to environmental factors, such as ultraviolet exposure, diet, and chemical compounds in paints, plastics, and other consumer products. Scientists suspected that the DNA polymerase was inserting nucleotides that were damaged by carrying an additional oxygen atom, he said, but past studies have not been able to demonstrate this process.

After the DNA polymerase (gray molecule in background) inserts a damaged nucleotide into DNA, the damaged nucleotide is unable to bond with its undamaged partner. Image credit: NIEHS

“When one of these oxidized nucleotides is placed into the DNA strand, it can’t pair with the opposing nucleotide as usual, which leaves a gap in the DNA,” said Dr. Wilson. “Until this paper, no one had actually seen how the polymerase did it or understood the downstream implications.”

Dr. Wilson and his colleagues observed the process in real time by forming crystal complexes made of DNA, polymerase, and oxidized nucleotides, and then capturing snapshots at different time points through time-lapse crystallography. The procedure not only uncovered the stages of nucleotide insertion, but indicated that the new DNA stopped the DNA repair machinery from sealing the gap. This fissure in the DNA prevented further DNA repair and replication, and in some cases even caused an immediate double-strand break.

“The damaged nucleotide site is akin to a missing plank in a train track,” said Dr. Wilson. “When the engine hits it, the train jumps the track, and all of the box cars collide.”

Large numbers of these pileups and double-strand breaks are lethal to the cell, serving as a jumping off point for the development of disease. However, this can be a good thing if you are a scientist trying to destroy a cancer cell.

“One of the characteristics of cancer cells is that they tend to have more oxidative stress than normal cells,” said Bret Freudenthal, Ph.D., lead author of the paper and postdoctoral fellow in Dr. Wilson’s group. “Cancer cells address the issue by using an enzyme that removes oxidized nucleotides that otherwise would be inserted into the genome by DNA polymerases. Research performed by other groups determined if you inhibit this enzyme, you can preferentially kill cancer cells.”

Dr. Wilson and Freudenthal stressed that the quantities of oxidized nucleotides in the nucleotide pool are usually under tight control, but if they accumulate and start to outnumber undamaged nucleotides, the DNA polymerase adds more of them to the strand. Molecules that inhibit oxidation, known as antioxidants, reduce the level of oxidized nucleotides, and may help prevent some diseases.